Project abstract: The number of long noncoding RNA (lncRNA) genes almost equals to the number of protein coding genes. However, the functions of most of lncRNA genes are unknown. Differential expression of lncRNA genes in different hematopoietic lineages implies that lncRNA genes are important regulators for hematopoiesis. One subgroup of lncRNA genes consists of antisense RNA genes that are transcribed in the opposite direction of their sense counterparts. Many genes important for hematopoiesis such as RBM15, PU.1, WT1, p53 and RUNX1 have antisense counterparts. In this proposed study, we plan to investigate the molecular details on how an antisense RNA (AS-RBM15) of RBM15 gene regulates hematopoiesis. We reported that AS-RBM15 promotes RBM15 protein translation via interaction with the 5'UTR of RBM15 mRNA. Furthermore, AS-RBM15 may promote megakaryocyte differentiation via RBM15-dependent and RBM15-independent pathways. We plan to address the hypothesis that AS-RBM15 regulates hematopoiesis via enhancing protein translation. 1) Determine how the cytoplasmic AS-RBM15 regulates RBM15 protein translation in hematopoietic cells. In this specific aim, we plan to understand how the RNA-RNA interaction involving AS-RBM15 and uORF in 5'UTR of RBM15 mRNA regulates RBM15 protein translation, and what kind of hematopoietic signals regulate AS-RBM15-mediated protein translation. 2) Determine the in vivo biological functions of mouse AS-Rbm15 in hematopoiesis. Although the primary RNA sequences between humans and mice are very different, the biological functions of AS-RBM15 genes may still be conserved. We plan to use mouse leukemia cell lines to investigate AS-Rbm15's molecular functions and to use bone marrow transplantation assays to investigate hematopoiesis in vivo. Accomplishing this proposal will provide us with new paradigms on RNA-mediated regulation, establish methods to study other antisense RNA genes and open up new avenues to understand the roles of long noncoding RNA in normal hematopoiesis.